U.S. patent application number 10/414942 was filed with the patent office on 2004-02-12 for preserved tissue matrix of a hollow organ, particularly of a blood vessel, a method of producing same, and the use thereof.
This patent application is currently assigned to co.don AG. Invention is credited to Lowel, Matthias, Siodla, Vilma.
Application Number | 20040029095 10/414942 |
Document ID | / |
Family ID | 28458952 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029095 |
Kind Code |
A1 |
Lowel, Matthias ; et
al. |
February 12, 2004 |
Preserved tissue matrix of a hollow organ, particularly of a blood
vessel, a method of producing same, and the use thereof
Abstract
The invention relates to a preserved, deantigenated tissue
matrix of an animal or human hollow organ, e.g. of a blood vessel,
of the ureter or urinary bladder, which matrix is autologous,
allogenic or xenogenic with respect to a recipient and whose
biomechanical properties are not or only slightly impaired by such
preservation, which does not include any infectious particles from
the donor and is excellently suited for coating with recipient
endothelial, epithelial, fibroblast, or muscle cells in order to
produce autologous grafts for said recipient. The invention is also
directed to a method of producing said tissue matrix and to the use
thereof, as well as to the autologous graft produced therefrom, and
to the production and use thereof.
Inventors: |
Lowel, Matthias; (Nurnberg,
DE) ; Siodla, Vilma; (Kleinmachnow, DE) |
Correspondence
Address: |
BRUCE LONDA
NORRIS, MCLAUGHLIN & MARCUS, P.A.
220 EAST 42ND STREET, 30TH FLOOR
NEW YORK
NY
10017
US
|
Assignee: |
co.don AG
Teltow
DE
|
Family ID: |
28458952 |
Appl. No.: |
10/414942 |
Filed: |
April 16, 2003 |
Current U.S.
Class: |
435/1.1 |
Current CPC
Class: |
A01N 1/0226 20130101;
A01N 1/02 20130101 |
Class at
Publication: |
435/1.1 |
International
Class: |
A01N 001/00; A01N
001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2002 |
DE |
102 17 779.1 |
Claims
1. A method for the production of a preserved tissue matrix of a
hollow organ, wherein a hollow organ tissue matrix collected from a
human or animal body, subsequent to optional mechanical removal of
adherent supporting, connective or fat tissue, is subjected to an
osmotic treatment by repeated washings with aqueous salt solutions,
the tissue matrix is deantigenated by repeated washings with
aqueous hydrogen peroxide solutions, the matrix is subsequently
treated with an alkaline solution and neutralized, the water in the
matrix is removed azeotropically by repeated washings with an
organic, water-miscible solvent, and the solvent-wet matrix is
dried.
2. The method according to claim 1, wherein 5 to 15% solutions,
preferably sodium chloride solutions, are used as salt solutions in
the osmotic treatment.
3. The method according to claim 1, wherein 3-10% H.sub.2O.sub.2
solutions are used.
4. The method according to claim 1, wherein a 0.1-1 N alkaline
solution, preferably NaOH or KOH, is used as alkaline solution.
5. The method according to claim 1, wherein ethanol is used as
organic solvent.
6. The method according to claim 1, wherein a preserved tissue
matrix of a blood vessel is produced, preferably of an artery or
vein.
7. The method according to claim 1, wherein a patch is produced
from the preserved blood vessel tissue matrix by axial
dissection.
8. A preserved tissue matrix of a hollow organ, produced according
to claim 1.
9. Use of a preserved tissue matrix according to claim 8 in the
production of a graft autologous with respect to the recipient by
rehydration of the matrix and colonization thereof with
recipient-specific cells.
10. Use of a preserved autologous tissue matrix according to claim
8 as a flat cover on defects in a human or animal body.
11. A method for the production of a graft autologous with respect
to the recipient by colonization of an autologous, allogenic or
xenogenic tissue matrix with recipient-specific cells, wherein a
preserved tissue matrix according to claim 8 is used, which matrix
is rehydrated, incubated with a suspension of recipient-specific
cells in a cell culture medium in a well-known fashion, and
cultured to achieve a surface completely coated with cells.
12. The method according to claim 11, wherein a preserved tissue
matrix of a blood vessel is used, preferably of an artery or
vein.
13. The method according to claim 11, wherein a preserved tissue
matrix of an axially dissected blood vessel (patch) is used.
14. The method according to claim 11, wherein the preserved tissue
matrix, subsequent to rehydration and prior to coating thereof with
cells, is coated with cell adhesion-mediating agents, preferably
with fibrin, fibrinogen or autologous serum.
15. The method according to claim 11, wherein the tissue matrix is
colonized with endothelial, epithelial and/or muscle cells from the
recipient.
16. An autologous graft, produced according to claim 11.
17. Use of an autologous graft according to claim 16 in abdominal,
thoracic, vascular or cardiac surgery, in urology or gynecology.
Description
[0001] The invention relates to a preserved, deantigenated tissue
matrix of an animal or human hollow organ, e.g. of a blood vessel,
of the ureter or urinary bladder, which matrix is autologous,
allogenic or xenogenic with respect to a recipient and whose
biomechanical properties are not or only slightly impaired by such
preservation, which does not include any infectious particles from
the donor and is excellently suited for coating with recipient
endothelial, epithelial, fibroblast, or muscle cells in order to
produce autologous grafts for said recipient. The invention is also
directed to a method of producing said tissue matrix and to the use
thereof, as well as to the autologous graft produced therefrom, and
to the production and use thereof.
[0002] In the course of operations on hollow organs of the blood
vessel, lymphatic, respiratory and urogenital systems, there is
frequently a need of replacing affected endogenous tissue and organ
structures.
[0003] Where sufficient replacement tissue of a patient (autologous
tissue) is available, as is the case e.g. in coronary bypass
surgery, the attending physician will preferably transplant the
autologous tissue.
[0004] Where autologous tissue is not sufficiently available, the
physician has the following options:
[0005] use of so-called biocompatible synthetic implants which may
be both absorbable and non-absorbable;
[0006] use of allogenic grafts, e.g. cardiac valves, blood
vessels;
[0007] use of xenogenic grafts, e.g. cardiac valves.
[0008] The use of synthetic implants sometimes involves severe side
effects such as foreign body reactions, allergic reactions, lacking
remodeling behavior, and also, life-threatening side effects such
as thromboses. For this reason, their use in e.g. vascular and
cardiac surgery is only possible in exceptional cases.
[0009] Apart from possible risks of infection, the use of allogenic
vital grafts is limited due to graft rejection.
[0010] Allogenic non-vital grafts (produced e.g. by
cryopreservation) have been used successfully for a long time in
replacing affected bone and connective tissue structures (e.g.
duraplasty, hernia operations).
[0011] However, their use in replacing tissue or organ structures
assuming important metabolic functions, such as blood vessels, is
limited. Lacking metabolic activity of the blood vessel walls gives
rise to complications such as thromboses.
[0012] Non-vital xenogenic grafts, such as chemically modified
cardiac valves of pigs, have been used successfully in cardiac
surgery for a long time. However, they also involve some drawbacks
such as calcification and lacking remodeling.
[0013] It is an object of the invention to provide a preserved
tissue matrix of a hollow organ, whose biomechanical properties are
largely the same as those prior to preservation, which is
deantigenated, does not include any infectious particles from the
donor, and is suitable for coating with recipient endothelial,
epithelial, fibroblast, or muscle cells in order to be used in the
production of autologous grafts for said recipient.
[0014] Another object of the invention is to devise a low-cost
method of producing autologous grafts which can be used in
abdominal, thoracic, vascular or cardiac surgery, in urology or
gynecology.
[0015] The object of the invention is accomplished by means of a
method for the production of a preserved tissue matrix of a hollow
organ, which method is characterized in that a hollow organ tissue
matrix collected from a human or animal body, subsequent to
optional mechanical removal of adherent supporting, connective or
fat tissue, is subjected to an osmotic treatment by repeated
washings with aqueous salt solutions, the tissue matrix is
deantigenated by repeated washings with aqueous hydrogen peroxide
solutions, the matrix is subsequently treated with an alkaline
solution and neutralized, the water present in the matrix is
removed azeotropically by repeated washings with an organic,
water-miscible solvent, and the matrix, now including solvent, is
dried.
[0016] In the meaning of the invention, said tissue matrix of a
hollow organ is understood to be a preferably tubular piece of
tissue of a blood vessel, e.g. a piece of an artery, vein or of a
lymphatic vessel. According to the invention, however, it is also
possible to use ureters, urinary bladders, tracheae, bronchial
tubes, tissues of stomach and intestinal walls. The invention
relates to all those tissues assuming an important metabolic
function, such as blood vessels, and those including both collagen
and elastin, as well as muscle cells.
[0017] According to the invention, the collected tissue--optionally
after previous removal of adherent supporting, connective or fat
tissue--is washed repeatedly with aqueous salt solutions and
optionally stored therein overnight in order to destroy the donor
cells included in the matrix by osmosis. 5-15% salt solutions are
used to this end. Sodium chloride, sodium phosphate, sodium
sulfate, sodium hydrogen phosphate, sodium hydrogen sulfate, sodium
citrate are possible as salts. It is preferred to use sodium
chloride.
[0018] This is followed by deantigenation effected by washing with
aqueous hydrogen peroxide solutions. This is followed by optional
storage in an H.sub.2O.sub.2 solution which preferably is 3-10%. It
has been determined that the use of H.sub.2O.sub.2 results in a
reduction of the DNA content in the tissue matrix by up to 50%.
[0019] According to the invention, the tissue matrix is
subsequently treated with an alkaline solution, preferably with
NAOH or KOH. It is preferred to use a 0.1-1 N alkaline solution.
Depending on the concentration of the alkaline solution, it has
been found that it is possible to achieve a reduction of the
genuine DNA content in the tissue matrix by up to 80% (with a 0.1 N
alkaline solution) or up to 100% when using a 1 N alkaline
solution.
[0020] In the last step, following neutralization, the matrix is
subjected to mild drying by azeotropic removal of water from the
matrix, which is done by repeated washing with an organic,
water-miscible solvent, e.g. ethanol, methanol, propanol, acetone.
According to the invention, the use of ethanol is preferred. This
step of solvent preservation of tissue is a well-known method
described e.g. in DE 29 06 650.
[0021] The combined use of H.sub.2O.sub.2 and alkaline solution in
the course of tissue matrix preservation achieves decellularization
of the matrix and complete elimination of matrix DNA and infectious
particles. In particular, the method of tissue preservation
according to the invention provides a matrix wherein biomechanical
stability and tissue structure remain largely unchanged after
rehydration, so that a graft produced using said matrix has good in
vivo tissue compatibility and undergoes partial or complete
remodeling inside the body, i.e., is transformed into autologous
tissue.
[0022] One particular advantage of the tissue matrix produced
according to the invention is that colonization with autologous
cells can proceed in such a way that in some or all of the cases
the use of components promoting cell adherence, such as fibrin,
fibrinogen or autologous serum, is not required. This dramatically
reduces the cost and greatly simplifies the production of
autologous grafts. Moreover, collecting autologous serum is
problematic in some cases, e.g. in heart patients, because large
volumes of blood must be withdrawn from these per se weakened
patients in order to collect the serum.
[0023] Apart from the preserved tissue matrix produced according to
the invention, the invention is also directed to the use thereof in
the production of a graft autologous with respect to the recipient
by rehydration of the matrix and colonization thereof by
recipient-specific cells, wherein several different types of cells
can be used, e.g. endothelial, epithelial and/or muscle cells to
coat a blood vessel, e.g. muscle cells and endothelial cells. It is
possible to combine multiple types of cells lying one on top of the
other. However, if multiple layers of cells are to be coated,
previous coating of the matrix with agents promoting cell adherence
is recommended, the amount required being low owing to the way of
matrix preservation.
[0024] The autologous cells required for colonization (of the
tissue matrix produced according to the invention, which is
allogenic or xenogenic with respect to the recipient) are collected
from tissue biopsy material having structural and metabolic
properties identical or similar to those of the tissue structure to
be replaced. The cells obtained after disintegration of the tissue
are cultured in a well-known manner.
[0025] Expansion of the cells to a number corresponding to the
respective area to be colonized is effected using appropriate
cell-specific culturing media, with optional addition of autologous
serum.
[0026] Prior to colonization, the preserved tissue matrix is
rehydrated, preferably using isotonic buffer solutions, and
subsequently fixed in a suitable apparatus for colonization, if
necessary. Thereafter, uniform coating of the rehydrated tissue
matrix can be performed using an agent promoting cell adherence.
According to the invention, however, said coating is omitted. For
coating, the tissue matrix is subsequently incubated with a cell
suspension including a well-defined number of autologous cells,
said incubation being effected repeatedly, if necessary.
Thereafter, cell culturing required to achieve a completely coated
surface is effected in specific cell culture media. After
completion of cell culturing, the graft is washed with isotonic
buffer solutions and subsequently packed in an isotonic salt
solution to be ready for dispatch.
[0027] Owing to the advantageous properties of the tissue matrix
employed, the grafts produced according to the invention, which
also represent a subject matter of the present invention, are
excellently suited for use in abdominal, thoracic, vascular or
cardiac surgery, in urology or gynecology.
[0028] Thus, in the event of a gastric tumor, for example, the
affected area can be replaced with a xenogenic patch colonized with
muscle and mucosa cells.
[0029] In tracheal partial resections, for example, the affected
section can be replaced with a xenogenic trachea colonized with
autologous epithelial cells.
[0030] Bypass operations in the peripheral and aortocoronary
regions can be performed using grafts consisting of allogenic
vascular matrices colonized with autologous endothelial cells.
[0031] In urinary bladder resection, a preserved hollow organ of
allogenic or xenogenic origin equal in size and colonized with
autologous epithelial cells can be transplanted as a
substitute.
[0032] According to the invention, it is also possible to produce a
patch from a preserved hollow organ tissue matrix by axially
dissecting the hollow organ, e.g. blood vessel. Following
rehydration, this piece of tissue, now being flat and colonized
with cells or not, can be used as a flat cover on defects in a
human or animal body.
[0033] For example, this is the case in the removal of cerebral
tumors and subsequent duraplasty using a patch not colonized with
cells.
[0034] Also, treatment of urinary incontinence using non-colonized
patches in a sling procedure is possible.
[0035] In this latter case, these patches optionally can be
colonized with autologous fibroblasts so as to achieve more rapid
tissue integration.
EXAMPLE
[0036] The following example exemplifies the production of a
coronary blood vessel consisting of a xenogenic matrix and coated
with autologous endothelial cells.
[0037] a) A vein about 15 cm in length and with an inner diameter
of about 4 mm is prepared from the muscular mass of a pig and
subsequently made free of adherent connective tissue by hand.
Thereafter, the lumen is washed repeatedly with a saline solution
until complete removal of blood is achieved. This is subsequently
washed with salt solutions so as to destroy the cells included in
the matrix by osmosis. This is followed by deantigenation with
hydrogen peroxide solutions. Thereafter, this is treated with 0.1 N
NaOH for one hour, followed by neutralization with acetic acid for
15 minutes and washing with purified water.
[0038] Azeotropic removal of water from the matrix thus obtained is
effected by repeated washing with ethanol.
[0039] The matrix thus treated is dried to a content of 50 ppm
ethanol at maximum and subsequently packed in a double-sterile
fashion. Final sterilization can be effected with gamma rays at a
dose of 25 kGy, for example.
[0040] b) The autologous endothelial cells are collected from a
suitable vein of the patient. Included blood components are removed
by repeated washing of the lumen with phosphate buffer solution.
Thereafter, the lumen of the vein is incubated with collagenase P
for about 20 minutes at 37.degree. C. The wash solution thus
obtained is centrifuged, the supernatant is discarded, and the
resulting cell pellet is resuspended in a cell culture medium
suitable for endothelial cells, e.g. Endomed.RTM.. Subsequently,
this cell suspension is seeded into a 25 cm.sup.2 cell culture
flask.
[0041] c) The endothelial cells are cultured for about 24 days at
37.degree. C., 95% r.h. and 5% CO.sub.2.
[0042] This includes two passages wherein transference into 75
cm.sup.2 and subsequently into 225 cm.sup.2 cell culture flasks is
effected.
[0043] d) The preserved pig vein is placed in an insulator through
a gate and rehydrated with 0.9% NaCl solution under aseptic
conditions. (Subsequently, the inner surface of the pig vein can be
coated with fibrin glue or autologous serum. Smoothing of the
fibrin glue or serum layer can be effected using a Fogarty
catheter.)
[0044] Subsequently, the pig vein thus pretreated is washed with
0.9% NaCl solution. Thereafter, the lumen is filled with
Endomed.RTM. endothelial cell medium having a well-defined number
of endothelial cells suspended therein. Both ends of the vein are
sealed by means of spring clamps. Now, the matrix thus treated is
placed in a Petri dish filled with Endomed.RTM. medium and
incubated for about 45-75 minutes.
[0045] Subsequently, this is transferred into a cell culture flask
and cultured at 37.degree. C. for 5-9 days, leaving the medium
unchanged.
[0046] e) After complete colonization of the pig vein with
endothelial cells, the Endomed.RTM. medium is removed, the pig vein
is washed with sterile phosphate buffer and subsequently filled
with 0.9% NaCl solution.
[0047] Shipping to physicians is effected in sterile packages in
0.9% NaCl solution at 2-8.degree. C. using a refrigerated
distribution chain.
* * * * *